CN102827297B - Homogeneous polysaccharide obtained by Ganoderma liquid submerged fermentation mycelium and preparation method thereof - Google Patents
Homogeneous polysaccharide obtained by Ganoderma liquid submerged fermentation mycelium and preparation method thereof Download PDFInfo
- Publication number
- CN102827297B CN102827297B CN201110156757.6A CN201110156757A CN102827297B CN 102827297 B CN102827297 B CN 102827297B CN 201110156757 A CN201110156757 A CN 201110156757A CN 102827297 B CN102827297 B CN 102827297B
- Authority
- CN
- China
- Prior art keywords
- homogeneous polysaccharide
- polysaccharide
- submerged fermentation
- ganoderma
- liquid submerged
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The open Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide of the present invention, it is characterised in that the total sugar content of described homogeneous polysaccharide is more than 95%, and the molecular weight of described homogeneous polysaccharide is more than 2000KDa.The present invention also discloses the preparation method of above-mentioned homogeneous polysaccharide.The polyoses content of the Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide that this method provides is higher, dosage is lower, purity is higher, and its preparation technology is also adapted to industrialized production.
Description
Technical field
The present invention relates to homogeneous polysaccharide GS-C-W-1, its preparation method and the purposes in preparing antitumor drug thereof obtained by Ganoderma liquid submerged fermentation mycelium.
Background technology
Edible and medicinal fungi has the multiple efficacies such as anti-tumor virus, blood fat reducing, slow down aging, protecting liver and expelling toxin, promotion nucleic acid and Protein synthesis, in state-owned long use history.Numerous studies show both at home and abroad, and fungus polysaccharide is isolated from fungus sporophore, mycelium, fermentation liquid, for the active component of fungal drug, are to control cell division differentiation, the class active polysaccharide that regulation cell growth is old and feeble.
Ganoderma (Ganodermasinense) is as Ganoderma fungus (2005 editions Chinese Pharmacopoeias), and it has obvious antitumor, improves effect of immunity.Owing to its preparation technology is indefinite, polyoses content relatively low (general ＜ 60%).Additionally Ganoderma crude polysaccharides complicated components, main pharmacodynamics position and component not yet illustrate.And due to Ganoderma sporophore resource scarcity, rare Ganoderma product on market.
Ganoderma crude polysaccharides dissolubility is very poor, and viscosity is relatively big, and color is deep, highly seasoned, and its molecular weight is from thousand of to up to ten million, and molecular weight span is very big, and difficulty finds suitable method of quality control.Research shows, molecular weight is one of principal element affecting polysaccharide component drug effect, if homogeneous polysaccharide can be obtained by isolated and purified for crude polysaccharides, illustrates its structure activity relationship, can not only improve drug effect level, and be very helpful quality control.
A kind of method that Chinese patent 200710045369.4 " a kind of Ganoderma fermentation process and prepared G.japonicum mycelium " discloses Ganoderma liquid submerged fermentation aerial mycelium, this mycelium decoction and alcohol sedimentation technique extracts the crude polysaccharides content obtained 39～75%, in its internal pharmacological evaluation, oral dose is to have certain anti-tumor activity when 40mg/kg/ days.Chinese patent 200710045368.x " intra-polysaccharides from mycelia of ganoderma sinensis and its preparation method and application " discloses a kind of method extracted from the mycelium that Ganoderma liquid submerged fermentation produces and prepare crude polysaccharides.The decoction and alcohol sedimentation technique that the method relates to extracts and obtains crude polysaccharides content 39～75%.The dosage of its pharmacological evaluation is also 40mg/kg/day (administering mode: oral medicine feed).Yang Guohong, Yang Yifang, Jin Juandi (antitumor active site research [J] of Ganoderma liquid submerged fermentation. Chinese herbal medicine, 2008,39 (6): 877-880) the anti-tumor activity polysaccharide of Ganoderma liquid submerged fermentation is studied, extracting from G.japonicum mycelium and obtain intracellular polysaccharide, the dosage of its pharmacological evaluation is also 40mg/kg/ days (administering mode: oral medicine feed).Chinese patent 0910574:200910197329.0 " a kind of G.japonicum mycelium anti-tumor polysaccharide component GS-C and preparation thereof " discloses the preparation of the Ganoderma liquid submerged fermentation mycelium polysaccharides of certain molecular weight scope.But, above patent application and article are all not directed to the research in terms of polysaccharide molecular weight, purity and structure elucidation aspect and mechanism of drug action.
In view of the situation of prior art, need the lower G.japonicum mycelium polysaccharide component of the crude polysaccharides that searching polyoses content is high, have obvious anti-tumor activity and dosage obtains than conventional method extraction.
Summary of the invention
The technical problem to be solved in the present invention is to provide the G.japonicum mycelium anti-tumor polysaccharide component that a kind of polyoses content is high, anti-tumor activity becomes apparent from and dosage is lower.
As used in the present invention, " liquid submerged fermentation " is a kind of fermentation process conventional in fermentation industry, and its culture medium is liquid.
As used in the present invention, " mycelium " is that mycelia gathers together and constitutes certain macrostructure.
As used in the present invention, " homogeneous polysaccharide " is the polysaccharide that monosaccharide passes through to be polymerized, and the sugar of polymerization has specific construction unit.The heteropolysaccharide that the polysaccharide that homogeneous polysaccharide can be divided into identical monosaccharide to be formed is formed with different monosaccharide.
First purpose of the present invention is to provide a kind of Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide, by its named GS-C-W-1, it is characterised in that the total sugar content of described homogeneous polysaccharide is more than 95%, preferably greater than 97.1%；And the molecular weight of described homogeneous polysaccharide is more than 2000KDa.
One according to the present invention preferred embodiment, and in homogeneous polysaccharide GS-C-W-1 of the present invention, the molecular weight of polysaccharide is more than 2000KDa.
One according to the present invention is preferred embodiment, analyzes it is known that the polysaccharide in described homogeneous polysaccharide GS-C-W-1 is mainly made up of galactose and rhamnose through TLC and HPLC, and described galactose is 3.3-3.7: 1 with the mol ratio of glucose, and preferably 3.5: 1.
Further, learning through periodate oxidation and Smith degradation analysis, the main chain that described homogeneous polysaccharide is connected by (1 → 6)-a-galactose is consisted of 1 → 6 rhamnose branch being substituted on main chain with having.
One according to the present invention preferred embodiment, and described homogeneous polysaccharide pH value in water is 7.16.
Second object of the present invention is to provide a kind of Ganoderma liquid submerged fermentation mycelium extract, and it comprises above-mentioned homogeneous polysaccharide, and the total sugar content of described homogeneous polysaccharide is more than 95%, and preferably 97.1%, and the molecular weight of described homogeneous polysaccharide is more than 2000KDa.
The present invention by different molecular weight separation of polysaccharides, thus obtains Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide GS-C-W-1 by column chromatography, and this homogeneous polysaccharide GS-C-W-1 can show certain drug effect on the premise of reducing dosage and not increasing toxicity.
Column chromatography can be according to different principles by isolated and purified, for the purpose of obtaining one-component for complicated mixture.And isolated and purified it is critical only that selected filler, one according to the present invention preferred embodiment, two fillers selected by the present invention are anion exchange resin and polydextran gel respectively, the former is to realize according to the difference by separation component ion-exchange capacity separating, and the latter separates according to the coil dimension by separation component molecule.
Third object of the present invention is to provide the preparation method of above-mentioned Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide GS-C-W-1, it is characterized in that, comprise the steps: that Ganoderma deep liquid mycelium crude polysaccharides, through column chromatography, obtains Ganoderma deep liquid mycelium homogeneous polysaccharide GS-C-W-1.
Above-mentioned preparation method is simple, with short production cycle, lower cost.
One according to the present invention preferred embodiment, and column chromatography steps is carried out by adding negative pressure.The instrument and equipment taked is peristaltic pump, automatic collection instrument.
One according to the present invention preferred embodiment, and two fillers selected by the present invention are anion exchange resin and polydextran gel respectively.
A particularly preferred embodiment according to the present invention, the filler anion exchange resin that column chromatography selects is DEAE-cellulose52.
A particularly preferred embodiment according to the present invention, the model of the filler polydextran gel that column chromatography selects is SephacrylS-300.
The present invention prepares Ganoderma liquid submerged fermentation mycelium polysaccharides component GS-C (obtaining according to method described in Chinese patent 0910574:200910197329.0 " a kind of G.japonicum mycelium anti-tumor polysaccharide component GS-C and preparation " thereof) that specifically comprises the following steps that of Ganoderma deep liquid mycelium homogeneous polysaccharide GS-C-W-1, and, through anion exchange resin, sephadex column obtains Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide GS-C-W-1.
Ganoderma liquid submerged fermentation mycelium crude polysaccharides purification is become homogeneous polysaccharide, pharmacological evaluation to show by profit column chromatography method of the present invention, and homogeneous polysaccharide GS-C-W-1 after purification has the activity of obvious enhancing human body immunity effect.
The Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide GS-C-W-1 content that the inventive method obtains is for more than 95%, and preferably 97.1% (phend-sulphuric acid detection), its molecular weight is more than 2000KDa.This homogeneous polysaccharide position is carried out pharmacological evaluation, GS-C-W-1 has the effect of highly significant at low concentrations to the T lymphocyte proliferation activity promoted, during LPS inducing mouse spleen B lymphocyte proliferation is reacted, along with concentration raises, its proliferation activity can obtain certain raising, but after bringing up to finite concentration, the facilitation of propagation starts to weaken；Prompting GS-C-W-1 reaches antitumor action by improving body immunity.And have no the toxic reaction being administered animal.
Therefore, third object of the present invention is to provide above-mentioned Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide GS-C-W-1 preparing antitumor drug and the application improved in immunity medicine.
Compared with prior art, the polyoses content of Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide GS-C-W-1 that this method provides is higher, dosage is lower, purity is higher, and its preparation technology is also adapted to industrialized production.
Accompanying drawing explanation
Fig. 1 is the HPGPC collection of illustrative plates of the embodiment of the present invention 1 gained Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide GS-C-W-1.
Fig. 2 is embodiment 1 step 2 with HPGPC detection) in molecular weight distribution (MWD) result of w-1.
Fig. 3 is embodiment 1 step 2) in w-1 cross the elution curve of SephacryS-300.
Fig. 4 is embodiment 1 step 2 with HPGPC detection) in molecular weight distribution (MWD) result of w-1-S300 (15-25).
Fig. 5 is embodiment 1 step 2) in w-1-S300 (15-25) cross SephacryS-300 elution curve.
Fig. 6 is the TLC result after GS-C-W-1 hydrolysis, and wherein 1 is fructose, and 2 is fucose, and 3 is arabinose, and 4 is rhamnose, and 5 is glucose, and 6,7 is GS-C-W-1, and 8 is galactose, and 9 is xylose, and 10 is galacturonic acid, and 11 is mannose.
Fig. 7 A-7D is respectively water, galactose, rhamnose, the HPLC collection of illustrative plates of the prepared GS-C-W-1 of embodiment 1.
Fig. 8 is periodic acid standard curve.
The HPLC collection of illustrative plates of the GS-C-W-1 degraded sample that Fig. 9 A-9D is respectively water, erithritol, glycerol, embodiment 1 prepare.
Figure 10 is the uv-spectrogram of the GS-C-W-1 that embodiment 1 prepares.
Figure 11 is the infared spectrum of the GS-C-W-1 that embodiment 1 prepares.
Figure 12 is the GS-C-W-1 that embodiment 1 prepares1H-NMR collection of illustrative plates.
Detailed description of the invention
In order to be more fully understood that the present invention, it is illustrated by the following examples, but these embodiments are not construed as limiting the invention.
Embodiment 1: the preparation of Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide GS-C-W-1:
1) preparation of Ganoderma liquid submerged fermentation mycelium crude polysaccharides GS-C:
Described Ganoderma liquid submerged fermentation mycelium crude polysaccharides GS-C is obtained by method described in Chinese patent 0910574:200910197329.0 " a kind of G.japonicum mycelium anti-tumor polysaccharide component GS-C and preparation thereof ".Concrete grammar is as follows: Ganoderma strain (Ganodermasinense) is buied from Shandong.Plating medium is sterilization 30 minutes at 121 DEG C, cultivate 4-6 days for 28 DEG C.121 DEG C of sterilizations of shake-flask seed culture medium 30 minutes, inoculate in sterilizing room after cooling, and on the shaking table between 28 ± 1 DEG C of shaking flasks, concussion is cultivated 5-7 days, and the shake-flask seed of select is cultivated 5-6 days in entering fermentation tank, finally obtains Ganoderma liquid fermentation mycelium.Mycelium water extraction 1～3h, extracting solution concentrating under reduced pressure, add ethanol precipitation, sediment fraction is dried to constant weight, obtains crude polysaccharides.By crude polysaccharides deionized water dissolving, with the hollow cellulose ultrafilter membrane ultrafiltration that the material that molecular cut off is 30,000 is polyamide, pressure is 0.1-0.3mPa, solution flow rate keeps good transmitance to be advisable according to film, collect trapped fluid, with the NF membrane concentrated liquid that molecular cut off is 200, lyophilization, obtain G.japonicum mycelium polysaccharide more than 30,000 positions.Taking and retained polysaccharide by ultrafilter membrane, with water dissolution, loading molecular cut off is the bag filter of 300,000, by liquid in bag filter: outer liquid=1 of bag: the ratios with water dialysis of 25, changes water 1 time every 2 hours.HPGPC collection of illustrative plates detection dialysis-effect is combined, to no longer appearing polysaccharide with Phenol sulfuric acid procedure.Taking trapped fluid, concentrated frozen is dried, and obtains polysaccharide component GS-C.
2) column chromatography combine HPGPC detection:
2.1DEAE-cellulose52 cellulose chromatography separates:
Take GS-C2.5g, dissolve with a small amount of deionized water solution, centrifugation, by molten with the examination of a small amount of deionized water again for precipitation, centrifugation, 3 times repeatedly, merge supernatant.Take the DEAE-cellulose52 cellulose column balanced on supernatant, be washed with deionized water de-, collect 2 column volumes of eluent with 10mL/ pipe.Every 2～5, effective phend-sulphuric acid measures its absorption value, and does elution curve.Merge the main peak that water elution liquid elutes, be designated as w-1；
HPGPC detects:
Chromatographic condition: chromatographic column: TSK-GELGMPWxl, column temperature: 35 DEG C, flowing is ultra-pure water mutually, and flow velocity 0.3mL/min, detector is differential refraction detector.
Behavior with HPGPC detection w-1 main peak.Sample preparation becomes the aqueous solution of 5mg/mL.Analysis result is shown in accompanying drawing 2.
From accompanying drawing 2, this elution fraction is mainly made up of the polysaccharide that retention time is 19min and 22min peak, according to retention time-molecular weight logarithmic scale curve, it is known that this stream part w-1 mean molecule quantity is more than 50w.Therefore select SephacrylS-300 (molecular weight separating ranges: 30000-1500000) that w-1 is chromatographed repeatedly.
2.2 first time SephacryS-300 purification
Precision weighs w-1 sample 500mg, is dissolved in 5mL deionized water, centrifugal is repeated 2 times except insoluble matter (centrifugal, 10min, 5000rpm), sucts clear liquid with burette and slowly instills SephacrylS-300 along tube wall surrounding and separate.Being washed with deionized water de-, flow velocity is 0.5mL/min, collects 5mL/ pipe, collects the volume of 1.5-2 post bed.Detect absorbance A 490 every 3 pipes with phend-sulphuric acid, and do elution curve.See accompanying drawing 3.
Merge main peak, be designated as w-1-S300 (15-25), concentrate, lyophilizing.
The behavior of each main peak is detected with HPGPC.Sample preparation becomes the aqueous solution of 5mg/mL.Carry out the purity detecting that chromatographic condition is identical.Analysis result is shown in accompanying drawing 4.
2.3 second time SephacryS-300 purification:
Precision weighs w-1-S300 (15-25) sample 250mg, is dissolved in 5mL deionized water, and the centrifugal insoluble matter that removes (is centrifuged, 10min, 5000rpm), it is repeated 2 times, sucts clear liquid with burette and slowly instill SephacrylS-300 along tube wall surrounding and separate.Being washed with deionized water de-, flow velocity is 0.5mL/min, collects 5mL/ pipe, collects the volume of 1.5-2 post bed.Detect absorbance A 490 every 3 pipes with phend-sulphuric acid, and do elution curve.See accompanying drawing 5.As shown in Figure 5: 2 peaks occurs in elution curve, separating effect is obvious, collects main peak 25#-43#, concentrating under reduced pressure to proper volume, lyophilizing, weighs, obtain 160mg.Obtain this method Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide GS-C-W-1.
Embodiment 2: Phenol sulfuric acid procedure measures the polyoses content of Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide GS-C-W-1 of the present invention
Specifically comprise the following steps that
Mark product are prepared: accurately weighed dried glucose 25mg, it is dissolved to 100ml, it is made into 250ug/ml and marks product solution, take 0.05 respectively, 0.1,0.15,0.2,0.25,0.3mL is in test tube, add deionized water and supply 0.3mL, add 5% phenol 0.7mL respectively and fully shake up, rapidly join concentrated sulphuric acid 4mL, 50 DEG C, 40min water-bath, measure absorbance after cooling.
Blank preparation: take 0.6ml deionized water, adds 5% phenol 1.4ml, concentrated sulphuric acid 8ml, heating in water bath, cooling, makees blank solution.
Specification Curve of Increasing: liquid to be measured for standard and blank solution ultraviolet spectrophotometry are surveyed its absorption value at 488nm, and does standard curve.
Sample preparation: take GS-C-W-1 sample, accurately weighed 5mg, add and the deionized water of 10ml is prepared 0.5mg/ml solution, take 0.05ml in test tube, add water and supply 0.3ml, be sequentially added into phenol, concentrated sulphuric acid, heating in water bath, cooling by mark product liquid, make sample liquid.
Detection: testing sample ultraviolet spectrophotometry is surveyed at 488nm its absorption value, and the polyoses content recording GS-C-W-1 with standard curve is 97.1%.
Embodiment 3: demarcate the molecular weight of polysaccharide in Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide GS-C-W-1 of the present invention by dextran standard Dextron series
Concrete grammar is as follows:
Chromatographic condition: TSK-GELGMPWxl chromatographic column；Flowing is water mutually；Flow velocity: 0.3mL/min；Column temperature 35 DEG C；Detector is: Composition distribution.
Mark product Dextron, GS-C-W-1 sample is respectively with water dissolution, sample introduction.
Measure glucosan Dextron series and the retention time of GS-C-W-1, do standard curve with the molecular weight logarithm value of the retention time-Dextron series of Dextron, it is known that the molecular weight of GS-C-W-1 is more than 2000KDa.
Embodiment 4: the structure elucidation of Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide GS-C-W-1
1) complete acid hydrolysis:
Take sample 8mg and be dissolved in 2mol/L trifluoroacetic acid (TFA), inflated with nitrogen tube sealing, 110 DEG C of hydrolysis 4h, intraluminal fluid evaporated under reduced pressure, add 2mL methanol and dissolve and evaporated under reduced pressure, be repeated 3 times to eliminate trifluoroacetic acid, add 1mL deionized water solution and make sample dissolve.
TLC result after 1.1GS-C-W-1 hydrolysis
Take this sample of 0.05mL with fructose, fucose, arabinose, mannose, glucose, gala aldehydic acid, xylose, galactose, rhamnose for comparison monosaccharide point sample respectively, respectively with acetone: water=24: 1, n-butyl alcohol: ethyl acetate: isobutanol: acetic acid: water: pyridine=35: launch at 100: 60: 35: 30: 35, finally develop the color with aniline-phthalic acid, and toast 10-15min, observed result at 110 DEG C.See accompanying drawing 6.
Learnt by Fig. 6: 4, No. 8 with GS-C-W-1 at same straight line, therefore may determine that GS-C-W-1 exists galactose, rhamnose monosaccharide.
1.2HPLC detects polysaccharide monosaccharide component
Chromatographic column condition:
Liquid-phase chromatographic column: TYPEUG80 (4.6 × 250mm)
Software processing system: Minennium32 version GPC data processing system
Detector: Water2410 differential refraction detector
Flowing phase: acetonitrile: water=3: 1
Column oven: 35 DEG C of detector temperatures: 35 DEG C
Flow velocity: 1mL/min
HPLC testing result is shown in accompanying drawing 7A-7D.
Savor the mole of galactose and rhamnose according to mark, in conjunction with the peak area ratio of polysaccharide sample HPLC, may infer that galactose in polysaccharide GS-C-W-1 monosaccharide: rhamnose=3.5: 1 (mol ratio).Can prove further TLC point plate exists galactose and rhamnose monosaccharide.
2) periodate oxidation:
2.1 periodic acid standard curves:
30mmol/L periodic acid liquid is prepared: precision weighs sodium metaperiodate 322.08mg and is surely dissolved in 50mL volumetric flask.
The drafting of standard curve: take 7 250mL volumetric flasks and be sequentially added into sodium periodate solution 0.2,0.4,0.6,0.8,1,1.5,2mL, add deionized water constant volume.After placing 10min, using water as blank, with spectrophotography, surveying trap at the wavelength of 223nm, with sodium metaperiodate μm ol/L as abscissa, optical density value is that vertical coordinate makees standard curve.It is shown in Table 1 and accompanying drawing 8.
Table 1, variable concentrations periodic acid are in the trap of 223nm
2.2 Malaprade reactions:
Precision weighs GS-C-W-110mg in 10mL volumetric flask, is subsequently adding 15mmol/LNaIO4 and is settled to scale.It is placed on dark place reaction (4 DEG C), sampling interval time (0h, 2h, 24h, 48h, 72h......) 0.1mL, with distilled water diluting 250 times, with ultraviolet spectrophotometer, blank is made with distilled water, absorbance is surveyed, until absorbance is constant at 223nm wavelength.Adding ethylene glycol and terminate reaction (neutralizing remaining periodic acid), periodate oxidation completes.
2.3 with NaOH titration formic acid growing amount:
0.1mol/LNaOH volumetric solution is demarcated: precision weighs the Potassium Hydrogen Phthalate 0.6g of 105 DEG C of constant weights and is dissolved in the cold water that 50mL newly boiled, and phenolphthalein, as indicator solution, is titrated to the aobvious pink of solution.Finally recording NaOH solution concentration is 0.092mol/L.
Methyl red indicator is prepared: precision weighs 0.1004g, adds 0.05mol/LNaOH solution 7.4mL and makes dissolving, adds water and be diluted to 200mL, to obtain final product.
Take the above-mentioned oxidation solution of 2mL, add 1 C.I. 13020. and make indicator, titrate with 0.0092mol/LNaOH (demarcating with Potassium Hydrogen Phthalate), calculate formic acid growing amount.
2.4 experimental results:
2.4.1 light absorption value records A absorption value time constant is 0.295.It is 0.607 that periodic acid liquid compares initial A absorption value, and periodic acid liquid absorption value after 72h is unchanged.Therefore the 0.757mol of periodic acid and example reaction can be drawn.
2.4.2 finally calculating formic acid growing amount according to titration is 0.34mmol.
2.4.3 and periodic acid consumption higher than two times of formic acid growing amount, illustrate that existence is not produced the hexose residue 1 → 2,1 → 2 of formic acid, 6,1 → 4,1 → 4,6 of bondings by periodate oxidation；In formic acid growing amount display molecule 1 →, the hexose residue of 1 → 6 of bonding account for 34%, the saccharide residue in periodic acid consumption and formic acid growing amount display molecule all can be oxidized.
3) Smith degradation reaction
Solution dialysis (Mw=3500) after GS-C-W-1 periodate oxidation after being processed by ethylene glycol, distilled water is dialysed 48 hours.Concentrate, add NaBH4 and reduce overnight.Being neutralized to pH with glacial acetic acid is 6～7, dialysis, and decompression is steamed to about 2mL, carried out lyophilization.The sample of reduction is put in ampoule bottle, adds 2mL2mol/LTFA, fill N2Rear tube sealing, hydrolyzes 4h at 110 DEG C, and the solution decompression in reaction bulb is evaporated, and adds the methanol of 1～2mL, is evaporated, and in triplicate to eliminate the TFA of excess, the 1mL water dissolution of the sample after hydrolysis, is HPLC and investigates.
3.1 chromatographic conditions:
Liquid-phase chromatographic column: TYPEUG80 (4.6 × 250mm)
Software processing system: Minennium32 version GPC data processing system
Detector: Water2410 differential refraction detector
Flowing phase: acetonitrile: water=3: 1
Column oven: 35 DEG C of detector temperatures: 35 DEG C
Flow velocity: 1mL/min
Reference substance is erithritol, glycerol.
3.2 results: see accompanying drawing 9A-9D.
Can be learnt by accompanying drawing 9D: Smith degradation reaction product HPLC detects substantial amounts of glycerol, illustrates containing the of bonding that can produce glycerol, i.e. 1 →, 1 → 2,1 → 6,1 → 2,6；Do not detect erithritol, illustrate not contain the of bonding generating erithritol, i.e. 1 → 4,1 → 4,6；Each several part does not all detect saccharide residue, illustrates that molecule is by being constituted by the of bonding of periodate oxidation.Therefore the of bonding that supposition GS-C-W-1 is is main link structure with 1 → 6 of bonding.
4) uv-spectrogram: see accompanying drawing 10.
Take GS-C-W-1 sample and carry out ultraviolet full wavelength scanner, at 260nm and 280nm, have no absorption, it was demonstrated that without nucleic acid and protein.
5) infared spectrum:
Take 1mgGS-C-W-1 sample, carry out IR detection with pressing potassium bromide troche.See accompanying drawing 11.
GS-C-W-1 infrared spectrogram has polysaccharide characteristic absorption, 3438.7cm-1Broad peak be the stretching vibration of-OH.2925.8cm-1Peak is the C-H stretching vibration of sugar, 1635.3cm-1For the water absworption peak combined, 1384.4cm-1It is the C-H angle vibration of sugar, more than can determine whether that GS-C-W-1 is polysaccharide compound.GS-C-W-1 is at 1074.8cm-1The characteristic absorption peak that peak is pyranoid ring, at 837cm-1There is absworption peak at place, and at 890cm-1Place is without absworption peak, and it may be the absworption peak of α-D galactopyranose, illustrates that GS-C-W-1 glycosidic bond type is α-configuration.
6) nuclear magnetic spectrum:
Take polysaccharide sample 10mg and load nuclear magnetic tube, be dissolved in heavy water, nuclear magnetic resonance analyser is carried out1H-NMR, spectrum analysis.
1H-NMR atlas analysis: visible C1 chemical shift of proton is positioned between δ 4.8～5.3 from accompanying drawing 12, then prove GS-C-W-1 type pyranohexose Han α, be consistent with infrared results.
Embodiment 5: the pharmacologically active of Ganoderma liquid submerged fermentation homogeneous polysaccharide GS-C-W-1 that embodiment 1 prepares
1) the anti-tumor in vivo experiment of GS-C:
1.1 animals: Kunming mouse, male
1.2 experimental techniques:
Take well-grown rat liver cancer H22 ascites or Lewis lung cancer tumor mass, with normal saline with 1: 4 dilution (cell concentration about 1-2 × 107Individual/ml), every mice right axil subcutaneous vaccination 0.2ml.
After inoculation, next day plays administration, and being administered volume is 0.5ml/20g body weight, the continuous lumbar injection of rat liver cancer H22 7 days, the continuous lumbar injection of Lewis lung cancer 10 days.Sunrecome per os gavage.Inoculating latter 10 days and dissect H22, within 14th, dissect Lewis lung cancer, take tumor mass, claim tumor weight, result judges according to below equation:
1.3 experimental results:
Table 1, the GS-C suppression ratio to mice H22
Compare with matched group:*P ＜ 0.05,**P ＜ 0.01.
Table 2, the GS-C suppression ratio to mice H22 and Lewis
Compare with matched group:*P ＜ 0.05,**P ＜ 0.01.
By table 1, table 2 understands the molecular weight polysaccharide position more than 300,000 (GS-C) stronger antitumor action.
2) the external MTT experiment of GS-C-W-1:
2.1 experiment materials and preparation:
(1) cell strain: A549 (human lung carcinoma cell line), SKOV3 (human oophoroma cell line), K562 (human leukemia cell line), SMMC-7721 (human hepatoma cell strain).Above-mentioned cell strain is studied pharmacological room by Shanghai medical industry and is preserved, and passes on maintenance.
(2) DMEM complete medium: add 10% new-born calf serum (GIBCOBRL), dual anti-；
(3) 0.25% trypsin solutions (Trypsin): purchased from Invitrogen company ,-20 DEG C of preservations.
(4) phosphate buffer (PBS): NaCl8g, KCl0.2g, Na2HPO41.15g, KH2PO40.2g, is dissolved in 1L distilled water, 121 DEG C of autoclave sterilization 20min, 4 DEG C of preservations.
(5) MTT (AMRESCO) solution: be made into 5mg/ml solution with PBS.
(6) lysate: every 100ml deionization distilled water is containing SDS10g, isobutanol 5ml, concentrated hydrochloric acid 0.1ml.
(7) reference substance: taxol BristolMyersSquibbSRL. lot number: 0E60359.
2.2 experimental techniques:
(1) prepared by sample and reference substance: be dissolved in PBS or DMSO by sample, obtains the solution that concentration is 10mg/ml.Make gradient dilution with PBS again, obtain concentration and be respectively 1000 μ g/ml, 100 μ g/ml, 10 μ g/ml, 1 μ g/ml, 0.1 μ g/ml, the dilute sample of 0.01 μ g/ml.Reference substance stock solution is 6mg/ml, makees gradient dilution with PBS, obtains concentration and is respectively 1000 μ g/ml, 100 μ g/ml, 10 μ g/ml, 1 μ g/ml, 0.1 μ g/ml, the dilute sample of 0.01 μ g/ml.
(2) sample diluted and reference substance are added in flat 96 orifice plates, every hole 10 μ l, makees two parallel testings at every.
(3) taking and be in the cell of exponential phase, cell is suspended in the DMEM culture medium containing 10% serum through trypsinization and after washing, and regulates cell suspending liquid density to 2 × l05Cell/ml.
(4) in flat 96 orifice plates, every hole adds 90 μ l cells, and last hole adds 90 μ l culture medium, in 37 DEG C, 5%CO2Cell culture incubator is cultivated 48 hours.
(5) every hole adds 20 μ l5mg/mlMTT solution, continue to be incubated 3～4 hours in incubator.
(6) every hole adds 100 μ l lysates, continues incubated overnight in incubator, makes the first crystal of generation fully dissolve.Measure 570nm absorbance value.
(7) by the computed in software sample IC50 to each tumor cell.
2.3 experimental results:
As seen from the above table: GS-C-W-1 does not has cytotoxicity to SKOV3, SMMC-772l, A549 human tumor cell line.
3) the ion vitro immunization experiment (research of the mouse spleen lymphocyte proliferation activity test of ConA and LPS induction of GS-C-W-1.)
3.1 animals: ICR mice, male and female do not limit.
3.2 experimental techniques:
3.2.1 reagent configures:
L-DMEM complete medium: configure according to a conventional method.
MTT solution: being made into 5mg/mL solution, 4 DEG C of preservations with PBS, two weeks interior effective.
ConA: being made into 10ug/mL solution by serum-free L-DMEM culture medium, 0.22um filtering with microporous membrane is degerming.
LPS: being made into 40ug/mL solution by serum-free L-DMEM culture medium, 0.22um filtering with microporous membrane is degerming.
3.2.2 prepared by splenocyte:
Mice is lethal with dislocation of cervical vertebra, the aseptic spleen that takes, and Ficoll separating Morr. cell is washed three times with L-DMEM culture fluid, counting, is made into 2 × 10 with the L-DMEM culture fluid containing 10% hyclone6Cell/mL cell suspension.
3.2.3 mouse spleen lymphocyte proliferation experiment:
By mouse boosting cell suspension o'clock on 96 well culture plates, every hole 100uL, if a matched group, add ConA or LPS (final concentration of 2.5ug/mL or 10ug/mL) of 50uL, it is eventually adding different amounts of GS-C-W-1 (final concentration of 10,50,100ug/mL), every hole culture fluid is supplied as 200uL.Tissue Culture Plate is placed in containing 5%CO2, 37 DEG C of incubators are cultivated.After 72h, add MTT solution (5mg/mL) 10uL, be placed in 5%CO2, 37 DEG C of incubators continue cultivate 4h.Taking out reactant, centrifugal (2000rpm, 5min), abandon supernatant, every hole adds 150uLDMSO, fully vibrates, and after grumeleuse is completely dissolved, on enzyme mark analyzer, 570nm reads A value.
3.3 experimental results:
In vitro the test result of the impact that mouse spleen lymphocyte converts is shown in Table 3, table 4.
Table 3, GS-C-W-1 are external on the ConA lymphopoietic impact of inducing mouse splenic T
Compared with matched group,*P ＜ 0.05,**P ＜ 0.01.
The impact of the external mouse spleen B lymphocyte proliferation on LPS induction of table 4, GS-C-W-1
Compared with matched group,*P ＜ 0.05.
Table 3 and table 4 result display GS-C-W-1 have the effect of highly significant at low concentrations to the T lymphocyte proliferation activity promoted, during LPS inducing mouse spleen B lymphocyte proliferation is reacted, along with concentration raises, its proliferation activity can obtain certain raising, but after bringing up to finite concentration, the facilitation of propagation starts to weaken；Prompting GS-C-W-1 reaches antitumor action by improving body immunity.
Claims (9)
1. Ganoderma liquid submerged fermentation mycelium homogeneous polysaccharide, it is characterized in that, the total sugar content of described homogeneous polysaccharide is more than 95%, described homogeneous polysaccharide is made up of galactose and rhamnose, and the molecular weight of described homogeneous polysaccharide is 3.3-3.7: 1 more than 2000KDa, described galactose with the mol ratio of rhamnose.
Homogeneous polysaccharide the most according to claim 1, it is characterised in that the total sugar content of described homogeneous polysaccharide is more than 97.1%.
Homogeneous polysaccharide the most according to claim 1, it is characterised in that described galactose is 3.5: 1 with the mol ratio of rhamnose.
4. according to the homogeneous polysaccharide described in Claims 2 or 3, it is characterised in that the main chain that described homogeneous polysaccharide is connected by (1 → 6)-a-galactose is consisted of 1 → 6 rhamnose branch being substituted on main chain with having.
5. according to the arbitrary described homogeneous polysaccharide of Claims 1-4, it is characterised in that described homogeneous polysaccharide pH value in water is 7.16.
6. Ganoderma liquid submerged fermentation mycelium extract, it comprises the arbitrary described homogeneous polysaccharide of claim 1-5, and the total sugar content of described homogeneous polysaccharide be more than 95%, and the molecular weight of described homogeneous polysaccharide is more than 2000KDa.
Extract the most according to claim 6, it is characterised in that the total sugar content of described homogeneous polysaccharide is more than 97.1%.
8. the preparation method of the arbitrary described homogeneous polysaccharide of claim 1-5, it is characterized in that, comprise the steps: that Ganoderma liquid submerged fermentation mycelium crude polysaccharides obtains homogeneous polysaccharide through column chromatography, filler used by described column chromatography steps is anion exchange resin and polydextran gel, described filler anion exchange resin is DEAE-cellulose52, and the model of described polydextran gel is SephacrylS-300.
9. the arbitrary described homogeneous polysaccharide of claim 1-5 is preparing antitumor drug and the application improved in immunity medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110156757.6A CN102827297B (en) | 2011-06-13 | 2011-06-13 | Homogeneous polysaccharide obtained by Ganoderma liquid submerged fermentation mycelium and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110156757.6A CN102827297B (en) | 2011-06-13 | 2011-06-13 | Homogeneous polysaccharide obtained by Ganoderma liquid submerged fermentation mycelium and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102827297A CN102827297A (en) | 2012-12-19 |
| CN102827297B true CN102827297B (en) | 2016-08-03 |
Family
ID=47330628
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110156757.6A Expired - Fee Related CN102827297B (en) | 2011-06-13 | 2011-06-13 | Homogeneous polysaccharide obtained by Ganoderma liquid submerged fermentation mycelium and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102827297B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103880970A (en) * | 2012-12-20 | 2014-06-25 | 上海医药工业研究院 | Method utilizing ion-exchange resin to decolor crude polysaccharide in ganoderma sinensis submerged fermentation mycelium |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102040748A (en) * | 2009-10-16 | 2011-05-04 | 上海医药工业研究院 | Ganoderma sinensis mycelium anti-tumor polysaccharide component GS-C as well as preparation method and application thereof |
-
2011
- 2011-06-13 CN CN201110156757.6A patent/CN102827297B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102040748A (en) * | 2009-10-16 | 2011-05-04 | 上海医药工业研究院 | Ganoderma sinensis mycelium anti-tumor polysaccharide component GS-C as well as preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 紫芝多糖的纯化和组分分析;叶姜瑜等;《西南师范大学学报》;20021231;材料与方法部分、结果与讨论部分、表1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102827297A (en) | 2012-12-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105175575B (en) | A kind of ganoderma lucidum beta glucan and its production and use | |
| CN102584963B (en) | Active Ganoderma lucidum polysaccharide peptide reference substance, and preparation method and application thereof | |
| CN109651532B (en) | Dendrobium officinale glucomannan | |
| CN107011453B (en) | A kind of dimension medicine just ancient polysaccharide of fiber crops and its extracting method and application | |
| CN103724445A (en) | Preparation method and blood sugar lowering function of Grifola frondosa polysaccharide F2 | |
| CN103239467B (en) | Agaricus blazei polysaccharide and preparation method thereof | |
| CN105859903A (en) | Radix glehniae polysaccharide and preparation method and application thereof | |
| CN110551230A (en) | Preparation method of astragalus polysaccharide | |
| CN107033253B (en) | A kind of purple dendrobium polysaccharide and its preparation and application | |
| CN102827297B (en) | Homogeneous polysaccharide obtained by Ganoderma liquid submerged fermentation mycelium and preparation method thereof | |
| CN106749729A (en) | A kind of Smilacina japonica polysaccharide and its preparation method and application | |
| CN102827298B (en) | Purple sesame liquid submerged fermentation mycelium homogeneous polysaccharide and preparation method thereof | |
| CN109682903A (en) | A kind of detection method of astragalus root polysaccharide molecular weight | |
| CN102276754B (en) | Organosulfate glucan in hedysarum polybotys saccharide as well as preparation method and application thereof | |
| CN103374078B (en) | Purple sesame liquid deep layer fermenting mycelium homogeneous polysaccharide and preparation method thereof and application | |
| CN104877037B (en) | Separation and purification method, products and application of Christia vespertilionis polysaccharides | |
| CN102040748B (en) | Ganoderma sinensis mycelium anti-tumor polysaccharide component GS-C as well as preparation method and application thereof | |
| CN108892732B (en) | Preparation method and application of Jinchang jujube polysaccharide with immunoregulation function | |
| CN109206532B (en) | Method for extracting, separating and purifying polysaccharide from myrtle fruits | |
| CN108948223A (en) | Hill gooseberry's polysaccharide P1 and its separation method and preparing the application in blood lipid-lowering medicine | |
| CN101744770B (en) | Ginseng polysaccharide injection and preparation method thereof | |
| CN102040749B (en) | Ganoderma sinense mycelium polysaccharide refined substance as well as preparation method and use thereof | |
| CN112220798B (en) | Application of oyster mushroom polysaccharide selenoside-III in preparation of medicine for treating ovarian cancer | |
| WO2014173056A1 (en) | Trametes robiniophila polysaccharide protein, preparation method therefor, and application thereof | |
| CN109134678B (en) | Polysaccharide P4, separation and purification method thereof and application of polysaccharide P4 in hypolipidemic drugs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160803 Termination date: 20200613 |